Microbial Examination of Non-Sterile Raw Materials and Products

Microbial Examination of Non-Sterile Raw Materials and Products Purpose

Microbial Examination of Non-Sterile Raw Materials and Products, To confirm that the bacterial & fungal count in the non sterile products & raw materials are within the In-house / Pharmacopoeia specification & free from certain microorganisms indicated in Pharmacopeia.

Scope

This SOP is applicable for microbiological test of Non-sterile Products such such as Oral Liquid, Semi-solid, Solid preparations and Raw Materials in Microbiology Section.

Definitions

Microbial Examination: Microbial examination is designed to determine the microbial contamination in non-sterile   products intended for Oral liquid, Topical Preparations or other non-sterile applications & Raw Materials.

• CSDA: Casein Soyabean Digest Agar
• CSDM: Casein Soyabean Digest Medium
• SDA  : Sabouraud Dextrose Agar
• SDB  : Sabouraud Dextrose Broth
• TAMC : Total Aerobic Microbial Count
• TYMC: Total Yeast & Mould Count

Responsibilities

The roles and responsibility is as follows:

Laboratory Attendant

Preparation Room for Microbiological Test

Microbiologist

Perform the test and incubation and in time proper documentation

Asst. Manager/Manager, Microbiology

Confirm test, incubation, report checking, document preservation and application of precise technical information.

Head of Quality Assurance

Take initiative regarding approval of this SOP

Procedure

Personal Precautions

• During enter into the test area, wear sterile gloves, Lab coat and eye protection (if necessary).
• To prevent unauthorized contamination, make sure that all personal ornaments, cell phone are left before entrance into the test room. The use of all type of Cell phone in the test area is strictly prohibited.
• Don’t move forcefully into the test area. Move always gently.

General Requirements for the test

Glass Apparatus:

• Pipette 2 ml, 10 ml
• Sterilized 90 mm Glass Petridish
• Screw capped Conical Flask 100 ml
• Screw Capped Test Tube
• Volumetric Flask 500 ml
• Volumetric Flask 1000 ml

Media and Reagents:

• Casein Soyabean Digest Agar(CSDA)
• Casein Soyabean Digest Broth(CSDB)
• Cetrimide Agar
• Mac-Conkey Broth
• MacConkey Agar
• Mannitol Salt Agar
• Meat peptone
• Neutralized Peptone
• Rapport Vasiliadis Salmonella Broth
• Sabouraud Dextrose Agar
• Xylose Lysine Deoxycholate(XLD) Agar

All of these media can be purchased from commercial available manufacturer.

Others Requirements

• 70% IPA or ethanol
• 0.45 µm Membrane Filter
• Filtration Unit(sterilized filter disk and filtering funnel)
• Forceps
• Glass spreader
• Scissors
• Surgical Gloves
• Surgical Cotton

Types of Test for Microbiological Examination

Enumeration Method (TAMC &TYMC)

This test quantify enumeration of mesophilic bacteria & fungi which may grow under aerobic 

Condition.

Test Conditions

• Wear latex free gloves, Head gear, mask and beard cover [if required], before enter into Test Room
• Use 70% IPA or ethanol to disinfectant the hands, the outer surface of test sample, LAF workstation with before start test.
• Perform the test under LAF to avoid contamination.
• Monitor the test area microbiologically with the help of Microbial Air Sampler at each working day.

Culture Media Preparation

• Prepare the different culture media as per specific requirements.
• Weigh the exact amount stated in the manufacturer label into right flask.
• Bring to boil completely to dissolve the media properly.
• Sterilize at 121⁰C for 15 minutes or as directed by the Manufacturer label.
• Store the prepared culture media in air tight flask properly at controlled environment.
• Store the prepared agar media at 2-8⁰C.
• Preserve the dehydrated culture media up to expiry date.
• Never use the expired culture media.
• Use the agar media when the temperature reduce near at 45⁰C & cool in case of the broth media.

Stock Buffer Solution

• Place 34 g of Potassium Dihydrogen Phosphate[KH₂PO₄] in a 1000 ml volumetric flask
• Dissolve in 500 ml of purified water, adjust to pH [7.2 ± 0.2] & dilute to 1000ml with purified water.
• Dispense  90 ml into  each screw capped flask 
• Sterilize at 121⁰C for 15 minutes.
• Store the prepared buffer at 2-8⁰C for a validated period.

Glassware Cleaning & Sterilization

• Initially clean all glassware by 1% detergent & then rinse with sufficient tap water.
• Finally Rinse with sufficient Purified Water to remove the residual content of detergent.
• Sterilize glassware at 200⁰C for 1 hour.

Testing of Products

Sample Size

• Collect 10 g or 10 ml of the products to be taken. 10 containers of the products from a batch.
• Collect the amount is not less than the amount present in 10 dosage units or 10 g or 10 ml of the respective product,  if amount per dosage unit is less than or equal to 1 mg.
• Take 1% of the batch size when batch size is less than 1000 ml or 1000 gm.
• Take 2 units or 1 units if the batch size is less than 100.

Types of Method

• Membrane Filtration
• Most Probable Number Method
• Pour Plate Method
• Surface spread Method

Membrane Filtration Method

• Prepare sample as per Method Suitability.
• Filter the sample through 0.45 µm & transfer the filter to the surface of CSDA for bacterial count and SDA[Sabouraud Dextrose Agar] for fungal count.
• Incubate CSDA[Casein Soyabean Digest Agar] at [30-35]⁰C for 3-5 days & at [20-25]⁰C for 5-7 days.
• After incubation, calculate the number of the cfu per gm or ml of the product.

Negative Control

Use diluents in place of test preparations. There must be no sign of growth in negative control. If found any sign of growth in negative control, the test must be invalid and repeat the whole test accordingly.

Pour Plate Method

• Prepare the sample as per the Method Suitability
• Pour 1 ml of prepared sample into the four 90 mm petridish, Add 15-20 ml CSDA[Casein Soyabean Digest Agar] into two plate & SDA into the others two plate.
• Allow to solidify & invert all plates.
• Incubate CSDA at [30-35]⁰C for 3-5 days and at [20-25]⁰C for 5-7 days.
• After incubation, calculate the number of the cfu per gm or ml of the product.

Negative Control

Use diluents in place of test preparations. There must be no sign of growth in negative control. If found any sign of growth in negative control, the test must be invalid and repeat the whole test accordingly.

Surface spread Method

• Prepare the sample as per Method Suitability.
• Spread not less than 0.1 ml of sample on the surface of two CSDA[Casein Soyabean Digest Agar]  and two SDA[Sabouraud Dextrose Agar] Plate.
• Dry all plates at Laminar Air Flow.
• Incubate the CSDA at [20-25]⁰C for 5-7 days and at [30-35]⁰C for 3-5 days.
• After incubation, calculate the number of the cfu per gm or ml of the product.

Negative Control

Use diluents in place of test preparations. There must be no sign of growth in negative control. If found any sign of growth in negative control, the test must be invalid and repeat the whole test accordingly.

Interpretation of the results

Count as Total Yeast/ Mould Count (TYMC) on SDA plate and Total Aerobic Microbial Count(TAMC) in CSDA plate and The acceptable criterion for microbiological quality is prescribed as :

• 101 cfu : maximum acceptable count =20
• 102 cfu : maximum acceptable count =200
• 103 cfu : maximum acceptable count =2000

Declaration

• The Material/product is passed when the observed count is less than specified count of that Material/product.
• The Material/product is failed if the observed count is greater than specified count of that Material/product.
• In that case, repeat the test, if the count is greater than specified count, the product is failed.

Test for Specified Microorganisms

Suitability of Test Method

Cary out the test in presence of the product. Add each test strain distinctly not more than 100 cfu at the time of product mixing with the culture media.The test will be suitable if found growth of the specific microorganism. The test will not suitable if no growth found the specific microorganism. In thatcase, add any neutralizer or increase the dilution for removal any inhibition of product.

Testing of Products

Test for E. coli

• Add 10 g or 10 ml of test sample to the 90 ml of Casein Soyabean Digest Medium. Incubate at [30-35]⁰C for 18-24 hours.
• Shake the container then transfer 1 ml of CSDM to the 100 ml of MacConkey Broth. Incubate at [42-44]⁰C for 24 hours.
• Sub-culture on MacConkey Agar plate from MacConkey broth. Incubate at [30-35]⁰C for 18-72 hours.
• The product complies with the test for E. coli if no red colonies are present with precipitated zone and the biochemical tests found negative[-ve].

Test for Salmonella

• Add 10 g or 10 ml of test sample to the 90 ml of Casein Soyabean Digest Medium. Incubate at [30-35]⁰C for 18-24 hours.
• Shake the container; transfer 0.1 ml of CSDM to 10 ml of RVS [Rappaport Vassiliadis Salmonella] Broth. Incubate at [30-35]⁰C hours for 18-24 hours.
• Sub-culture on XLD [Xylose Lysine Deoxycholate] Agar plate from RVS [Rappaport Vassiliadis Salmonella] Broth . Incubate at [30-35]⁰C for 18-48 hours.
• The product complies with the test for Salmonella if no red colonies are present with or without black centres and the biochemical tests are negative[-ve].

Test for Pseudomonas aeruginosa

• Add 10 g or 10 ml of test sample to the 90 ml of Casein Soyabean Digest Medium. Incubate at [30-35]⁰C for 18-24 hours.
• Sub-culture on Cetrimide Agar plate from CSDM [Casein Soyabean Digest Medium]. Incubate at [30-35]⁰C for 18-72 hours.
• The product complies with the test for Ps. aeruginosa if no bluish green colonies are present and the biochemical tests are negative[-ve].

Test for C. albicans

• Add 10 g or 10 ml of test sample to 90 ml of SDB [Soubaurad Dextrose Broth]. Incubate at [30-35]⁰C for 3-5 days.
• Sub-culture on SDA[Soubaurad Dextrose Agar] plate from [Soubaurad Dextrose Broth]. Incubate at 30-35⁰C for 24-48 hours.
• The product complies with the test for C. albicans if no white colonies are present and the   biochemical tests are negative[-ve].

Test Report Preparation

• Report the result in Microbial Count Report of Non-sterile RM, Annexure-I.
• Report the result in Microbial Count Report of Non-sterile Products, Annexure-II.

This is all about the Microbial Examination of Non-Sterile Raw Materials and Products and based on this information you can generate a SOP for Microbial Examination of Non-Sterile Raw Materials and Products.

Download all Annexure

Annexure I Microbial Count Report of Non-Sterile Raw Materials

Annexure II Microbial Count Report of Non-Sterile Products

Annexure III Non Sterile Raw Materials Log book

Annexure IV Non Sterile Products Log book